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Clinical & Experimental Metastasis

, Volume 27, Issue 1, pp 55–69 | Cite as

Protease inhibitor SERPINA1 expression in epithelial ovarian cancer

  • Karine Normandin
  • Benjamin Péant
  • Cécile Le Page
  • Manon de Ladurantaye
  • Véronique Ouellet
  • Patricia N. Tonin
  • Diane M. Provencher
  • Anne-Marie Mes-MassonEmail author
Research Paper

Abstract

Epithelial ovarian cancer is the most lethal gynecologic cancer with a 5 years survival rate of 30–40% in patients diagnosed with high-grade invasive disease (TOV). This is in stark contrast to the 95% 5 years survival rate in ovarian cancer patients diagnosed with low malignant potential (LMP) disease. The progression from localized tumor to invasive metastasis involves matrix proteolysis. Protease inhibitors are thought to play a key role by limiting this process. Using the Affymetrix HG-U133A GeneChip array, we have studied all serine protease inhibitors and found several serpin family members that are differentially expressed between LMP and TOV serous tumors. SERPINA1 was selected for further study due to its high expression in the majority of LMP tumors and its low expression in TOV tumors; observations that were also validated by quantitative-PCR (Q-PCR). To study the effects of its over expression on different tumorigenic parameters, SERPINA1 was cloned in the pcDNA3.1+ plasmid which was subsequently used to derive stable clones from two invasive ovarian cancer cell lines, TOV-112D and TOV-1946. We found no effect of SERPINA1 over expression on tumor growth in SCID mice although cell migration and invasion were affected in in vitro assays. There was also no association between patient survival and SERPINA1 immunostaining, however, SERPINA1 localization was different in LMP (nuclear) and TOV (cytoplasmic) tumors. SERPINA1 remains an interesting candidate since protein homeostasis, regulated by proteases and their inhibitors, should be studied holistically in order to assess their full impact in tumor progression.

Keywords

Biological effects Epithelial ovarian cancer Ovarian cell lines Protease inhibitor SERPINA1 

Abbreviations

AAT/SERPINA1

Alpha 1-antitrypsin/Serpin peptidase inhibitor, clade A, member 1

BOV

Benign ovarian tumor

DAB

Diaminobenzidine

ELISA

Enzyme-linked immunosorbent assay

ERK1

Extracellular signal-regulated kinase 1

FBS

Fetal bovine serum

FIGO

International Federation of Gynecology and Obstetrics

G

Tumor grade

ICC

Intraclass correlation coefficient

LMP

Low malignant potential tumor

OSE

Ovarian surface epithelium

ROC

Receiver Operating Characteristic

SCID

Severe combined immunodeficiency

S2N

Signal-to-noise ratio

TOV

Invasive ovarian tumor

Notes

Acknowledgments

We are grateful to Jason Madore, Kim Leclerc Desaulniers, Liliane Meunier, Lise Portelance, Marie-Line Puiffe, and Louise Champoux for technical assistance. We would like to thank laboratory members for thoughtful discussion, and Luke Masson, Jason Madore and Jennifer Kendall-Dupont for careful reading of the paper. We are also grateful to the members of the Department of Gynecologic Oncology and the Department of pathology of the CHUM for providing specimens. This research was supported by grants from Génome Canada/Génome Québec and the Canadian Institutes of Health Research (CIHR) to Anne-Marie Mes-Masson, Patricia N. Tonin, and Diane M. Provencher. Tumor banking was supported by the Banque de tissus et de données du Réseau de recherche sur le cancer des Fonds de la Recherche en Santé du Québec (FRSQ), affiliated to the Canadian Tumor Repository Network (CTRNet). Karine Normandin was supported by studentships from the Canderel Fund of the Institut du cancer de Montréal and the molecular biology department of the Faculté des études supérieures de l’Université de Montréal. Véronique Ouellet was supported by studentships from the CIHR and the Canderel Fund of the Institut du cancer de Montréal.

Supplementary material

10585_2009_9303_MOESM1_ESM.ppt (50 kb)
Supplementary Table 1 Clinical characteristics of tumors used for RT-Q-PCR analysis. No patient received chemotherapy prior to the surgery. (PPT 49 kb)
10585_2009_9303_MOESM2_ESM.ppt (236 kb)
Supplementary Figure 1 SERPINA1 mRNA and protein expression in TOV-112D and TOV-1946 derived clones. a Expression of SERPINA1 mRNA in transfected clones. After 30 rounds of SERPINA1 and ERK1 amplification, PCR products were separated on a 2% agarose gel. ERK1 was used as a reference gene. b Overexposure of low level expressing clones. Total protein extracts (50 μg) were separated on a 10% polyacrylamide gel and transferred onto PVDF membrane. Equal loading was evaluated using a β-actin antibody. (PPT 236 kb)
10585_2009_9303_MOESM3_ESM.ppt (194 kb)
Supplementary Figure 2 High variability of tumor formation in SCID mice injected with TOV-1946 derived clones. (PPT 193 kb)
10585_2009_9303_MOESM4_ESM.ppt (7.5 mb)
Supplementary Figure 3 In situ expression of SERPINA1 in ovarian tissues. a Specificity of SERPINA1 antibody tested by immunocytochemistry on TOV-1946 over expressing SERPINA1 cells and the parental TOV-1946 cell line. b Staining of both cell lines (TOV-112D and TOV-1946); and the different subcellular localizations of SERPINA1 in LMP and TOV tumors obtained by immunohistochemistry on the ovarian cancer tissue microarray. (PPT 7721 kb)
10585_2009_9303_MOESM5_ESM.ppt (617 kb)
Supplementary Figure 4 Cellular morphology of the SERPINA1 over expressing clone S112D-1, the TOV-112D cell line, and the control clone C112D-B. (PPT 617 kb)

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Karine Normandin
    • 1
  • Benjamin Péant
    • 1
  • Cécile Le Page
    • 1
  • Manon de Ladurantaye
    • 1
  • Véronique Ouellet
    • 1
  • Patricia N. Tonin
    • 4
    • 5
  • Diane M. Provencher
    • 1
    • 2
    • 3
  • Anne-Marie Mes-Masson
    • 1
    • 3
    • 6
    Email author
  1. 1.Institut du cancer de MontréalCentre de recherche du centre hospitalier de l’Université de Montréal (CHUM)MontrealCanada
  2. 2.Division of Gynecologic OncologyUniversité de MontréalMontrealCanada
  3. 3.Department of MedicineUniversité de MontréalMontrealCanada
  4. 4.Department of Human GeneticsThe Research Institute of McGill University Health CenterMontrealCanada
  5. 5.Department of MedicineThe Research Institute of McGill University Health CenterMontrealCanada
  6. 6.CR-CHUM/ICMMontrealCanada

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